WGS of S. cerevisiae: recombinant offspring of BY4741xRM11.1a FACS sorted for extreme high / low UPS activity
We used the Nextera DNA library kit (Illumina, San Diego, CA, USA) according to the manufacturers instructions with the following modifications. For the tagmentation reaction, 5 ng of genomic DNA from each sample was diluted in a master mix containing 4 uL of Tagment DNA buffer, 1 uL of sterile molecular biology grade water, and 5 uL of Tagment DNA enzyme diluted 1:20 in Tagment DNA buffer. The tagmentation reaction was run on a SimpliAmp thermal cycler (Thermo Fisher Scientific) using the following parameters: 55 C temperature, 20 uL reaction volume, 10 minute incubation. To prepare libraries for sequencing, we added 10 uL of the tagmentation reaction to a master mix containing 1 uL of an Illumina i5 and i7 index primer pair mixture, 0.375 uL of ExTaq polymerase (Takara Bio, Mountain View, CA, USA), 5 uL of ExTaq buffer, 4 uL of a dNTP mixture, and 29.625 uL of sterile molecular biology grade water. We generated all 96 possible index oligo combinations using 8 i5 and 12 i7 index primers. The library amplification reaction was run on a SimpliAmp thermal cycler with the following parameters: initial denaturation at 95 C for 30 seconds, then 17 cycles of 95 C for 10 seconds (denaturation), 62 C for 30 seconds (annealing), and 72 C for 3 minutes (extension). We quantified the DNA concentration of each reaction using the Qubit dsDNA BR assay kit (Thermo Fisher Scientific) and pooled 10 L of each reaction. This pooled mixture was run on a 2% agarose gel and we extracted and purified DNA in the 400 bp to 600 bp region using the Monarch Gel Extraction Kit (NEB) according to the manufacturers instructions.     We submitted pooled, purified DNA libraries to the University of Minnesota Genomics Center (UMGC) for Illumina sequencing. Prior to sequencing, UMGC staff performed three quality control (QC) assays. Library concentration was determined using the PicoGreen dsDNA quantification reagent (Thermo Fisher Scientific) with libraries at a concentration of 1 ng/L passing QC. Library size was determined using the Tapestation electrophoresis system (Agilent Technologies, Santa Clara, CA, USA) with libraries in the range of 200 to 700 bp passing QC. Library functionality was determined using the KAPA DNA Library Quantification kit (Roche, Penzberg, Germany), with libraries with a concentration greater than 2 nM passing. All submitted libraries passed each QC assay. We submitted 7 libraries for sequencing at different times. Libraries were sequenced on a NextSeq 550 instrument (Illumina). Depending on the number of samples, we used the following output settings. For libraries with 70 or more samples (2 libraries), 75 bp paired end sequencing was performed in high-output mode to generate approximately  reads. For libraries with 50 or fewer samples (5 libraries), 75 bp paired end sequencing was performed in mid-output mode to generate approximately  reads. Average read coverage of the genome ranged from 9 to 35 with a median coverage of 28 across all libraries. Sequence data de-multiplexing was performed by UMGC.